The location of neurons generating the rhythm of breathing in mammals is unknown. By microsection of the neonatal rat brainstem in vitro, a limited region of the ventral medulla (the pre-Bötzinger Complex) that contains neurons essential for rhythmogenesis was identified. Rhythm generation was eliminated by removal of only this region. Medullary slices containing the pre-Bötzinger Complex generated respiratory-related oscillations similar to those generated by the whole brainstem in vitro, and neurons with voltage-dependent pacemaker-like properties were identified in this region. Thus, the respiratory rhythm in the mammalian neonatal nervous system may result from a population of conditional bursting pacemaker neurons in the pre-Bötzinger Complex.